As one of medical diagnoses, a pathological diagnosis is performed. A pathologist diagnoses a disease using a tissue section taken from a human body and notifies a clinician of the necessity or unnecessity of a therapy or operation. On the basis of conditions of a patient and pathological diagnosis, a physician determines a drug treatment plan, and a surgeon determines whether or not an operation should be performed.
In pathological diagnosis, it is widely performed to prepare a tissue sample by slicing a tissue specimen obtained through resection of an organ or needle biopsy into a thickness of about several micrometers and observe an enlarged image of the tissue sample with an optical microscope for obtaining various findings. In many oases, the sample is prepared by fixing a taken tissue through dehydration and paraffin blocking, slicing it into a thickness of several micrometers, and removing the paraffin. Since the sample, which barely absorbs and scatters light, is substantially colorless and transparent, staining with a dye is usually performed prior to the observation.
As a staining method, various staining methods have been proposed.
In particular for the tissue sample, hematoxylin-eosin staining (HE staining) using two dyes, hematoxylin and eosin, is typically used as a morphological observation staining for observing the morphology of the sample (Non-Patent Document 1 and Patent Documents 1 and 2).
The hematoxylin stains cell nuclei, calcareous sites, cartilage tissue, bacteria, and mucus to a color of from indigo blue to light blue. The eosin stains cytoplasm, interstitial tissue, various fibers, erythrocytes, and keratinocytes to a color of from red to deep red. A pathologist makes a diagnosis on the basis of morphological information and staining information such as changes in size and shape of cell nuclei and changes in tissue pattern in the microscopic image of the stained tissue sample.
As other staining for morphological observation, for example, Papanicolaou staining (Pap staining) used for cytodiagnosis is also known.
In pathological diagnosis, immunological observation called immunostaining where a molecular target is stained for confirming the expression of molecular information of a sample in order to diagnose dysfunction such as abnormal expression of a gene or a protein is performed.
The immunostaining, for example, employs dye staining with an enzyme (DAB staining). The DAB staining stains antigens as observation objects with antibodies modified so as to be stained by a dye, and measures the antigen level through observation. Alternatively, fluorescent labeling is employed. The fluorescent labeling stains antigens as objects with antibodies modified with a fluorescent dye and measures the antigen level through observation.
An attempt has currently been made to simultaneously perform morphological and immunological observations of a sample. For example, it has teen tried to perform HE staining for morphological observation and DAB staining for immunological observation simultaneously (Patent Document 3).
However, the staining with an enzyme label such as DAB staining develops a color similar to the color in HE staining to preclude the distinction between the staining by an enzyme label and the HE staining, which makes the simultaneous observation difficult. In addition, in DAB staining, the concentration of the stain considerably varies depending on environmental conditions such as temperature and time to preclude the estimation of the actual amount of, for example, antibodies on the basis of the concentration of the stain.
Meanwhile, a fluorescent label is used in pathological diagnosis.
The fluorescence method has superior quantitative characteristics compared with DAB staining (Non-Patent Document 1).
However, simultaneous performance of pathological diagnosis and morphological observation with fluorescent labels has a disadvantage in that results of the staining are readily affected by the fluorescence of the staining reagent used for the tissue staining. A possible countermeasure is to use an infrared excitation/emission fluorescent dye which is not affected by visible light (Patent Document 4). For example, infrared-emitting dyes such as Alexa Fluor 647 (Molecular Probes) and CY5 (GE Healthcare) are known.